Multidimensional analysis methods and apparatus are especially suitable for use with complex biochemical samples. An example of such multidimensional apparatus comprises two chromatographic columns in series, wherein the two columns are typically selected to cause separation of the components according to different physiochemical properties. For example, the first dimension column may be such as to separate components by ion pair chromatography while the second dimension column may be a reverse-phase analytical column. In such a system, components eluting from the first dimension column pass directly into the second dimension column where they are subjected to further separation before arriving at a detector.
Although most commonly used with liquid mobile phases and especially for HPLC, multidimensional chromatographic systems for gas chromatography or supercritical fluid chromatography, or combinations of all three, are known.
A limitation of the simple system wherein two or more columns are simply connected in series is that the same mobile phase must pass through both columns. Further, especially in the case of samples comprising many components, there is a danger that once a first batch of components has passed through the first dimension column and is undergoing separation of the second dimension column, some components comprised in a second batch eluting from the first dimension column may pass onto the second dimension column and interfere with the separation thereon of the first batch of components.
These problems can be overcome by interposing one or more traps between the first and second dimension columns. This allows the temporary storage of a second batch of components eluting from the first dimension column while a first batch is undergoing separation on the second dimension column. It also allows the use of different mobile phases for the first and second dimension separations. For example, by means of a suitable arrangement of valves, a first mobile phase may be employed to carry out a first dimension separation and to trap a batch of components, and a second mobile phase may be used to release the trapped components and to carry out a second dimension separation. In some cases, when the first mobile phase is such that it does not cause components to be eluted from the second dimension column, it may be possible to dispense with a separate trap and to trap batches of components at the head of the second dimension column before changing the mobile phase to one which will elute the trapped components.
Where provided, a suitable trap may comprise a short chromatographic column capable of trapping at least some of the components entering it in the mobile phase used for the first dimension separation. In some cases it may comprise a chromatographic absorbent similar to that used in the second dimension column, but other types of trap may also be used, for example traps based on affinity or immunological binding. Trapped components may be released from the trap when required by introduction of a different, stronger, mobile phase.
When a trap is provided downstream of the first dimension column, the second dimension column may be replaced by alternative analytical apparatus, for example a mass spectrometer. Using such apparatus, a typical method of analysis may comprise trapping at least some of the components as they elute from the first dimension column and subsequently releasing batches of them into the mass spectrometer for further analysis.
Quantitative calibration of multidimensional analysis systems is more difficult than it is for single dimension systems. For example, an internal standard added to a complex sample analysed on a multidimensional system will typically elute in only one or a few of the batches of sample components which elute from the first dimension column, and therefore can only provide a reference for those batches as they undergo further separation on the second dimension column (or analysis in analytical apparatus such as a mass spectrometer). Unfortunately, it is often impractical to overcome this difficulty by providing many different internal standards, each appropriate for the components of each batch eluting from the first dimension. Consequently, in the past, quantitative calibration of multidimensional chromatographic systems has often been carried out merely by separately running one or more samples comprising a known amount of a component whose concentration in a complex mixture is to be determined, using identical chromatographic conditions. The quantity of the component in the complex mixture may then be determined by comparing the heights or areas of the relevant chromatographic peaks obtained from the calibration sample and the mixture. Such a method is of course less accurate than calibration using an internal standard added to the sample itself because it relies on the chromatographic conditions being identical for the run of the calibration sample and the run of the mixture. In practice, in view of the long time periods typically required for multidimensional chromatography, and the complexity of the methods used, maintaining the conditions sufficiently similar for accurate quantification is very difficult. Other errors may also arise if the matrix in which the calibration sample is comprised is significantly different from that in which the sample is comprised. Use of a mass spectrometer to detect compounds eluting from the chromatographic system, commonplace when complex samples are being analysed, adds further to these quantification problems because its sensitivity can be affected by many other factors that may also vary with time and matrix composition.
Similar difficulties in quantification arise in systems where the second dimension column is omitted and eluent from the trap is fed directly to a mass spectrometer.
There is therefore a need for multidimensional analysis apparatus and methods in which the problems associated with quantitative calibration are minimized. It is an object of the present invention to provide such apparatus and methods. It is another object of the invention to provide methods and apparatus for quantitative multidimensional analysis that do not require internal standards to be added to a complex sample. It is further object to provide methods and apparatus for quantitative multidimensional analysis that do not rely on comparing chromatographic parameters obtained during different runs.
According to a first aspect, an embodiment of the invention provides a method of chromatography comprising the following steps:
First, a sample comprising one or more sample components is introduced into a first mobile phase, and the resulting solution is passed through first chromatographic media so that at least some of the sample components are retained on the first chromatographic media. Then, a releasing phase comprising calibration material is passed through the first chromatographic media. The releasing phase is such as to cause at least some of the sample components previously retained on the first chromatographic media to be released from that media into the releasing phase. The calibration material is such that it is not retained on the first chromatographic media in the presence of the releasing phase and may comprise one or more chemical species. The releasing phase comprising the released sample components and the calibration material is passed into second chromatographic media which is capable of separating at least some of the released sample components and the calibration material. The eluent from the second chromatographic media is passed into detector means. The detector means generates signals indicative of the amount of at least some of the sample components and the calibration material present in the eluent. These signals are then processed to yield quantitative information about at least some of the sample components relative to the calibration material.
In some embodiments, only some of the sample components retained on the first chromatographic media are released by the passage of the releasing phase. Once further separation of the released sample components on the second chromatographic media is completed (or sufficiently advanced to ensure there will be no interference), a second batch of sample components may be released from the first chromatographic media by passing another releasing phase into it. This releasing phase may also comprise calibration material, and will typically be stronger than the releasing phase originally used so that a second batch of sample components, more strongly retained on the first chromatographic media, may be released. The releasing phase comprising the second batch of sample components and the calibration material may then be passed into the second chromatographic media and at least some of the sample components therein may be quantitatively determined in the same way as described for the first batch of sample components.
A convenient way of generating different releasing phases is to provide a plurality of reservoirs, each containing a releasing agent as well as the calibration material. Each reservoir may contain a different concentration of releasing agent. The contents of a reservoir may be introduced into the flow of first mobile phase to generate a releasing phase as required. This may be done using the same apparatus provided for introducing a sample into the flow of first mobile phase, for example a sample valve and sample loop similar to those conventionally used in liquid chromatography. For example, if the first chromatographic separation media comprises a strong cation exchange media, the releasing agent may comprise salt solutions having different concentrations in different reservoirs.
The process may be repeated using still stronger releasing phases, each capable of releasing another batch of sample components retained on the first chromatographic media, and each releasing phase comprising calibration material that is not retained on the first chromatographic media. In this way calibration material will elute in every batch of sample components transmitted to the second chromatographic media, allowing quantitative calibration of the sample components. This overcomes the problem inherent in prior multidimensional methods wherein the calibration material elutes only with certain batches of sample components.
In a variation, the releasing phases may be modified by the addition of a modifying phase as they exit from the first chromatographic media, thereby generating a second mobile phase that passes to the second chromatographic media. The modifying phase may be selected so that the resultant second mobile phase is more suitable for carrying out the separation on the second chromatographic media than the releasing phases alone. The composition of the modifying phase may be varied with time, permitting gradient elution to be carried out. The flow of releasing phase may be discontinued once the sample components have all passed onto the second chromatographic media so that the additional solvent provides the sole source of mobile phase for the second chromatographic media. A splitting device may also be provided so that only a portion of the releasing phase and/or the modifying phase passes to the second chromatographic media.
It will be appreciated that in the above methods each of the releasing phases used to release sample components from the first chromatographic media enters the second chromatographic media and comprises at least a part of the second mobile phase used to carry out the separations on that media, at least for some of the time during which a separation is being carried out. In certain cases this may degrade the separation, or even be substantially incompatible with it, even if the releasing phase is modified as described. The invention therefore provides methods of quantitative multidimensional analysis having more general applicability. In these methods, batches of sample components released from the first chromatographic media are trapped on trapping media before being passed to a second analysis device. This may comprise a second chromatographic column or an analyzer such as a mass spectrometer. Thus the first mobile phase and/or releasing phases may be used to carry sample components from the first chromatographic media to trapping media rather than directly to the second chromatographic media or analyzer. The trapping media should be capable of trapping thereon at least some of the sample components in the presence of the first mobile phase or releasing phases eluting from the first chromatographic media. During this stage of the methods, the first mobile phase and/or releasing phases may be diverted to waste rather than being passed to the second separation media or analytical device. When a batch of sample components has been trapped on the trapping media, the flow of first mobile phase or releasing phase may be discontinued and a separate supply of a second mobile phase may be used to release at least some of them from the trapping media, to pass them to the second separation means or analytical device. Another separation may then be carried out on the second separation media, or an analysis carried out on the analytical device.
The trapping media may be comprised in a separate chromatographic column, and may have properties similar to those of the second chromatographic media where provided. Alternatively, the trapping media may comprise part of the second chromatographic media itself, so that sample components are trapped on the initial portion of the second chromatographic media itself, until the second mobile phase is introduced. This variation requires that the first mobile phase and releasing phases pass through the second chromatographic media and may not always be applicable.
In all cases, however, calibration material is provided in each of the releasing phases. The calibration material, which may comprise one or more chemical species, may be selected so that in the presence of the releasing phase it is either not retained or is only retained for a short time on the first chromatographic media, but is capable of being trapped on the trapping media along with the sample components. Calibration material may then be released by the second mobile phase and may pass with the sample components into the second chromatographic media or analysis device, thereby allowing at least relative quantitative determination of at least some of the sample components in that batch.
The use of intermediate trapping between the first chromatographic media and second chromatographic media or analysis device decouples the first and second dimension separation from the second dimension separation or analysis. This may allow a conventional chromatographic separation to be carried out on the first chromatographic media, in place of the trapping and subsequent batch releasing of sample components described above. In such a case, unresolved groups of sample components eluting from the first chromatographic media may be trapped on the trapping media, and subsequently released by a second mobile phase to pass into the analysis device or second chromatographic media where they may undergo further separation. An aliquot of calibration material may be separately introduced into the first mobile phase. The calibration material, which may comprise one or more chemical species, should not be retained, or be retained only for a short time, on the first chromatographic media, but should be retained on the trapping media along with the sample components. Alternatively, calibration material may be introduced into the second mobile phase.
Calibration material may be introduced into a mobile phase or a releasing phase by means of a sample valve configured to introduce a fixed volume of a solution comprising the calibration material into the flow of the phase. Alternatively, in embodiments where a fixed volume of releasing phase is used to release a batch of trapped sample components, each releasing phase may comprise calibration material. When a fixed volume of the releasing phase is injected into a mobile phase that in the absence of the releasing solvent is incapable of releasing any sample components from the chromatographic or trapping media, this method will introduce a fixed quantity of the calibration material. Typically, several different releasing phases of different strengths are prepared, each containing the calibration material. These different strength phases may then be introduced as required into the mobile phase to release different batches of sample components from the media.
In all the above embodiments, if an absolute quantitative determination of the sample components is required, each releasing phase should comprise a known amount of calibration material. However, if only relative quantification is required, it may only necessary for each releasing phase to comprise the same amount of calibration material.